Carbohydrate Research 338 (2003) 1895 /1905
www.elsevier.com/locate/carres
Note
Structure of the lipopolysaccharide of Pseudomonas aeruginosa O-12
with a randomly O-acetylated core region
Olga V. Bystrova,a,b Buko Lindner,b Herman Moll,b Nina A. Kocharova,a
Yuriy A. Knirel,a,b,* Ulrich Zähringer,b Gerald B. Pierc
a
N.D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Leninsky prospect 47, 119991 Moscow, Russia
b
Research Center Borstel, Center for Medicine and Biosciences, 23845 Borstel, Germany
c
Channing Laboratory, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115, USA
Received 28 April 2003; accepted 19 June 2003
Abstract
The lipopolysaccharide of Pseudomonas aeruginosa O-12 was studied by strong alkaline and mild acid degradations and
dephosphorylation followed by fractionation of the products by GPC and high-performance anion-exchange chromatography and
analyses by ESI FT-MS and NMR spectroscopy. The structures of the lipopolysaccharide core and the O-polysaccharide repeating
unit were elucidated and the site and the configuration of the linkage between the O-polysaccharide and the core established. The
core was found to be randomly O- acetylated, most O -acetyl groups being located on the terminal rhamnose residue of the outer
core region.
# 2003 Elsevier Ltd. All rights reserved.
Keywords: Lipopolysaccharide; Core structures; O-antigen repeating unit; O-acetylation; Pseudomonas aeruginosa
Pseudomonas aeruginosa is an opportunistic human
pathogen, which causes severe infections in hosts with
weakened defense mechanisms. Based on the immunospecificity of the O-antigens (O-polysaccharide chains of
the lipopolysaccharides), strains of P. aeruginosa are
classified into more than 20 O-serotypes (Ref. 1 and refs
cited therein).
The structure of the chemical repeating unit of the Opolysaccharide has been determined in all P. aeruginosa
O-serotypes.1,2 However, it could not be ascertained
from this structure what are the first and last monosaccharides in the repeating unit, nor which monosac-
Abbreviations: 8eLeg,
5,7-diamino-3,5,7,9-tetradeoxy-Lglycero -D-galacto -non-2-ulosonic acid (8-epilegionaminic
acid); Cm, carbamoyl; Etn, ethanolamine; FucN, 2-amino2,6-dideoxygalactose;
Hep,
L-glycero-D-manno -heptose;
HPAEC, high-performance anion-exchange chromatography;
Kdo, 3-deoxy-D-manno -oct-2-ulosonic acid; LPS, lipopolysaccharide; QuiN, 2-amino-2,6-dideoxyglucose.
* Corresponding author. Tel.: /7-095-9383613; fax: /7095-1355328.
E-mail address: [email protected] (Y.A. Knirel).
0008-6215/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0008-6215(03)00290-8
charide is linked to the LPS core. The chemical
repeating unit may be any cyclic permutation of the
actual biological repeating unit, that is the properly
ordered oligosaccharide, which, after having been preassembled on a lipid carrier, is polymerized into the Opolysaccharide in the O-antigen polymerase (Wzy)dependent pathway of LPS biosynthesis. Later, the
biological repeating unit has been defined in P. aeruginosa serotypes O-1, O-5, O-6, O-10, and O-11,3 7 and
the biosynthesis pathways of the O-antigens of P.
aeruginosa serotypes O-5 and O-6 have been discussed.8
The structure of the LPS core has been established in
P. aeruginosa strains belonging to various Oserotypes4 7,9 and in a number of rough mutants,9 12
including a cystic fibrosis isolate, P. aeruginosa 2192,
having the full core.11 Peculiar features of the core of P.
aeruginosa are a high degree of phosphorylation and the
presence of various non-sugar groups, including ethanolamine diphosphate, N-linked alanyl (or less commonly acetyl) and O-linked carbamoyl groups. In
addition, in some strains O -acetyl groups are present
in non-stoichiometric amounts in the outer core region
at multiple unknown positions.4,5,11
1896
O.V. Bystrova et al. / Carbohydrate Research 338 (2003) 1895 /1905
In this work, we determined the structure of the
biological repeating unit of the P. aeruginosa O-12
polysaccharide and demonstrated a random distribution
of the O -acetyl groups in the LPS core of this strain. The
structure of the chemical repeating unit of the Opolysaccharide of P. aeruginosa O-12 has been determined1,2,13 and found to be a linear trisaccharide that
contains 2-acetamido-2,6-dideoxy-D-glucose (D-QuiNAc), 2-acetimidoylamino-2,6-dideoxy-L-galactose (LFucNAm), and 5,7-diacetamido-3,5,7,9-tetradeoxy-Lglycero-D-galacto-non-2-ulosonic acid (di-N -acetyl-8epilegionaminic acid, 8eLeg5Ac7Ac14).
Strong alkaline degradation of the LPS following
mild hydrazinolysis resulted in a mixture of oligosaccharide phosphates, which was isolated by GPC on
Sephadex G-50 and then fractionated by HPAEC at
super-high pH (Fig. 1) to give two major fractions,
INaOH and IINaOH, and a minor fraction IIINaOH. The
isolated compounds were analyzed by ESI FT-MS and
NMR spectroscopy.
Fraction IINaOH was found to contain two core-lipid
A backbone oligosaccharide pentakisphosphates (1 and
3, Fig. 2) in the ratio /7:1, respectively, having the
same molecular mass 2357.51 Da. They showed an 1H
NMR spectrum (Table 1) practically identical to that of
the corresponding compounds isolated by HPAEC after
strong alkaline degradation of the LPS of P. aeruginosa
immunotype 4 (O-1).6 Compounds 1 and 3 are isomers
that differ in the position of the terminal rhamnose
residue in the outer core region (core glycoforms 1 and
2). In smooth P. aeruginosa strains studied,3 6 glycoform 1 core carries no O-polysaccharide, whereas glycoform 2 core is mainly (Ref. 6 and this work) or
completely3 5 substituted with an O-polysaccharide
chain.
Minor fraction IIINaOH contained one compound 2
having molecular mass 2211.46 Da. The mass difference
of 146.05 Da between compound 2 on the one hand and
compounds 1 and 3 on the other hand corresponds to
the lack of the rhamnose residue. This was confirmed by
the 1H NMR spectrum of 2, which showed only minor
signals for rhamnose. That 2 is not an artifact was
confirmed by ESI FT-MS of the products derived by
mild acid degradation of the LPS (see below). No
Fig. 1. HPAEC of the alkaline degradation products from the LPS of P. aeruginosa O-12. Fractions I, II and III correspond to the
core-lipid A backbone oligosaccharide with one O-antigen repeating unit, the unsubstituted core-lipid A backbone oligosaccharide,
and the core-lipid A backbone lacking Rha, respectively.
O.V. Bystrova et al. / Carbohydrate Research 338 (2003) 1895 /1905
1897
Fig. 2. Structures of the alkaline degradation products from the LPS of P. aeruginosa O-12, 1 /4. Abbreviations: 8eLeg, 5,7diamino-3,5,7,9-tetradeoxy-L-glycero -D-galacto -non-2-ulosonic (8-epilegionaminic) acid; FucN, 2-amino-2,6-dideoxygalactose;
Hep, L-glycero -D-manno -heptose; Kdo, 3-deoxy-D-manno -oct-2-ulosonic acid; QuiN, 2-amino-2,6-dideoxyglucose. All monosaccharides are in the pyranose form and have the D-configuration unless stated otherwise.
rhamnose-lacking compound was derived on alkaline
degradation under the same conditions from the LPS of
other P. aeruginosa strains studied earlier.4 6,11
Fraction INaOH contained two compounds 4a and 4b
in the ratio /5:1, which have molecular masses 2879.51
and 2921.52 Da, respectively. These values correspond
to the core-lipid A backbone oligosaccharide pentakisphosphates bearing one O-antigen repeating unit,
which is either fully N-deacetylated 4a or retains one
of the N -acetyl groups 4b. A similar incomplete Ndeacetylation was observed on strong alkaline degradation of the LPS of P. aeruginosa immunotype 4 (O-1).6
Compounds 4a,b were evidently derived from a shortchain SR-type LPS, which, together with long-chain Stype and O-antigen-lacking R-type LPS, is typically
present in P. aeruginosa .1
The structure of the core-lipid A backbone moiety in
4 (Fig. 2) was established as described previously4,5,11
based on the full assignment of the 1H NMR spectrum
(Fig. 3, Table 1) using 2D 1H,1H COSY and TOCSY
experiments followed by linkage and sequence analyses
by 2D 1H,13C HMQC and ROESY experiments and
determination of the phosphorylation pattern by a
1
H,31P HMQC experiment (Table 2). As a result, a
typical glycoform 2 core was identified in 4, which is
identical to that found in the LPS of the other P.
aeruginosa smooth strains studied.4 6
The structure of the O-antigen trisaccharide moiety in
4 (Fig. 2) was determined using a 2D ROESY experi-
ment, which revealed FucN H-1,QuiN H-3 and QuiN
H-1,Rha H-3 correlations at d 5.40/3.73 and 4.81/4.00 in
4a or d 5.25/3.80 and 4.72/3.86 in 4b, respectively. Based
on these data combined with the structure of the
chemical repeating unit of the O-antigen,13 it was
inferred that 8eLeg is attached at position 3 of FucN
and occupies the terminal non-reducing end of the Oantigen trisaccharide. The a configuration of the FucN
linkage and the b configuration of the QuiN linkage
were established by J1,2 values of 3 and 7 Hz, respectively. The a configuration of 8eLeg followed from the
axial orientation of the carboxyl group, as shown by a
relatively large difference of 0.81 ppm between H-3eq
and H-3ax chemical shifts.13 Compound 4b differs from
4a by a significantly lower-field position of the H-2
resonance of QuiN (d 3.93 vs. 3.05), which demonstrates
N-acetylation of QuiN in 4b.
The data obtained in this work and previously13,14
define the structure of the biological repeating unit and,
thus, the full structure 5 of the O-antigen of P.
aeruginosa O-12 (Fig. 4). As in the other D-QuiNAccontaining LPS of P. aeruginosa ,4 6 this sugar is the
first monosaccharide of the O-polysaccharide and is blinked to the terminal rhamnose residue of the glycoform 2 core. Therefore, the anomeric configuration of DQuiNAc in the first and interior O-12-antigen repeating
units is different, which is in accordance with the
involvement of different enzymes, O-antigen polymerase
(Wzy) and ligase (WaaL), with connection of the
1898
O.V. Bystrova et al. / Carbohydrate Research 338 (2003) 1895 /1905
Table 1
1
H NMR data of the alkaline degradation products from the LPS of P. aeruginosa O-12 (d )
Sugar residue
H-1
H-2
H-3
H-4
H-5
H-6(6a)
H-6b(7a)
H-7b
H-3ax
H-3eq
H-4
H-5
H-6
H-7
H-8(8a)
H-9(8b)
Compound 1
0/6)-a-D-Glcp NI-1-P
0/6)-b-D-Glcp NII-(1 0/
0/4,5)-a-KdopI-(2 0/
a-KdopII-(2 0/
0/3)-a-HeppI2,4P -(1 0/
0/3)-a-HeppII6P -(1 0/
0/3,4)-a-D-Galp N-(1 0/
0/6)-b-D-GlcpI-(10/
0/6)-a-D-GlcpII-(10/
a-D-GlcpIII-(10/
a-L-Rhap -(10/
5.67
4.88
2.02
1.82
5.39
5.19
5.61
4.68
5.05
4.99
4.81
3.44
3.08
2.22
2.10
4.55
4.42
3.84
3.26
3.51
3.58
4.03
3.93
3.87
4.12
4.17
4.21
4.25
4.49
3.57
3.77
3.71
3.83
3.64
3.76
4.31
4.09
4.49
4.16
4.41
3.27
3.59
3.47
3.46
4.17
3.77
3.69 a
3.73 a
4.35
4.02
4.24
3.73
4.19
3.69
3.75
3.79
3.47
3.88
4.15
4.01
4.49
3.89
3.82
3.79
3.79
1.33
4.32
3.72
3.64
3.81
3.73
3.75
3.91
3.91
3.91
3.86
Compounds 4a,b b
0/6)-a-D-Glcp NI-1-P
0/6)-b-D-Glcp NII-(1 0/
0/4,5)-a-KdopI-(2 0/
a-KdopII-(2 0/
0/3)-a-HeppI2,4P -(1 0/
0/3)-a-HeppII6P -(1 0/
0/3,4)-a-D-Galp N-(1 0/
0/3,6)-b-D-GlcpI-(10/
a-D-GlcpII-(10/
a-D-GlcpIII-(10/
0/3)-a-L-Rhap -(10/
0/3)-b-D-Quip N-(10/
0/3)-a-L-Fucp N-(10/
a-8eLegp -(20/
5.67
4.88
2.03
1.81
5.39
5.21
5.61
4.68
5.06
5.02
5.19 (5.15)
4.81 (4.72)
5.40 (5.25)
1.91
3.44
3.07
2.21
2.11
4.52
4.45
3.84
3.50
3.51
3.58
4.25
3.05 (3.93)
3.63 (3.46)
2.72
3.92
3.87
4.13
4.21
4.21
4.26
4.49
3.69
3.77
3.74
4.00 (3.86)
3.73 (3.80)
4.16 (4.19)
3.65
3.64
3.75
4.31
4.09
4.47
4.18
4.42
3.55
3.52
3.42
3.65 (3.51)
3.36 (3.39)
4.02 (4.00)
3.01
4.17
3.75
3.69 a
3.76 a
4.32
4.01
4.24
3.70
4.09
3.75
4.06 (4.01)
3.54
4.36 (4.37)
4.22
3.79
3.50
3.91
4.15
4.02
4.49
3.90
3.85
3.85
3.75
1.28 (1.26)
1.35 (1.33)
1.22
3.40
4.32
3.74
3.66
3.82
3.75
3.76
3.91
3.93
3.88
3.89
a
b
4.03
3.91
4.02
3.96
3.80
3.94
4.01
3.98
3.82
1.42 (1.41)
Tentative assignment.
When different, data of 4b are given in parentheses. The signal for the NAc group in 4b is at d 2.06.
repeating units to each other and transfer of the Opolysaccharide or a single O-antigen repeating unit to
the core, respectively.8
The LPS was cleaved by mild hydrolysis with aqueous
1% HOAc at 100 8C and a core oligosaccharide mixture
was fractionated by GPC on Sephadex G-50 to give two
fractions, IHOAc and IIHOAc, which correspond to the
core with one O-antigen repeating unit and an unsubstituted core, respectively. ESI FT-MS studies, using
published data on similar oligosaccharides from the LPS
of P. aeruginosa immunotype 5,5 showed that both
fractions were mixtures of oligosaccharides that differ in
the number of phosphate groups (P2-4) and O -acetyl
groups (Ac0-2) and in the presence or absence of
ethanolamine (Etn). There were present also minor
compounds that lack Rha and O-polysaccharide fragments consisting of one to four repeating units. The
major compounds in fraction IIHOAc were core oligosaccharides RhaGlc3(GalNAla)Hep(HepCm)KdoP4EtnAc0-1 with molecular masses 1892.44 and 1934.45 Da
for the non-acetylated compound 6 and its mono-O-
acetylated derivative, respectively, as well as the corresponding oligosaccharides with Kdo in an anhydro
form(s) (Fig. 5(B)). Molecular masses 2581.75 and
2623.77 Da of the major compounds in fraction IHOAc
(Fig. 5(A)) differ from those in fraction IIHOAc by 689.3
Da, which corresponds to one O-antigen repeating unit
(see structure 5 in Fig. 4).
A mixture of 6 and O -acetylated 6 was isolated from
fraction IIHOAc by HPAEC under neutral conditions.
The mixture was studied by NMR spectroscopy, including 2D 1H,1H COSY, TOCSY, 1H,31P HMQC and
1
H,31P HMQC-TOCSY experiments, as described by us
earlier in studies of the products that were derived in the
same way from the LPS of P. aeruginosa immunotype
5.5 The compounds from both strains were found to
share a number of structural features, including the
same phosphorylation pattern, the presence of an N alanyl group at GalN, an O -carbamoyl group at HepII,
and ethanolamine diphosphate (EtnPP ) at HepI (Fig.
6). The major distinction of 6 is the presence of three
glucose residues rather than four in the P. aeruginosa
O.V. Bystrova et al. / Carbohydrate Research 338 (2003) 1895 /1905
Fig. 3. 1H NMR spectrum of compounds 4a,b obtained by alkaline degradation from the LPS of P. aeruginosa O-12. Arabic numerals refer to protons in sugar residues. For
abbreviations see legend to Fig. 2. Designations for Rha, QuiN, FucN and 8eLeg in 4a and 4b are not italicized and italicized, respectively.
1899
1900
O.V. Bystrova et al. / Carbohydrate Research 338 (2003) 1895 /1905
Table 2
31
P NMR data of the alkaline degradation products from the
LPS of P. aeruginosa O-12 (d )
Sugar residue
Compound 1
0/6)-a-D-Glcp NI-1-P
0/6)-b-D-Glcp N4PII-(10/
0/3)-a-Hepp 2,4PI-(1 0/
0/3)-a-Hepp 6PII-(1 0/
Compounds 4a,b
0/6)-a-D-Glcp NI-1-P
0/6)-b-D-Glcp N4PII-(10/
0/3)-a-Hepp 2,4PI-(1 0/
0/3)-a-Hepp 6PII-(1 0/
P-1
P-2
P-4
1.89
3.87
4.18
P-6
3.41
4.60
1.94
0.38
3.04
3.04
3.88
Fig. 4. Full structure of the O-polysaccharide of the P.
aeruginosa O-12 LPS 5. Abbreviations: 8eLeg5Ac7Ac, 5,7diacetamido-3,5,7,9-tetradeoxy-L-glycero -D -galacto -non-2ulosonic (di-N -acetyl-8-epilegionaminic) acid; FucNAm, 2acetimidoylamino-2,6-dideoxygalactose; QuiNAc, 2-acetamido-2,6-dideoxyglucose.
immunotype 5 LPS.5 As expected based on the alkaline
degradation data of the LPS (see above), 6 represented
mainly core glycoform 1, which was contaminated by a
small amount (/15%) of the core glycoform 2 isomer.
The products from P. aeruginosa O-12 were distinguished also by O-acetylation to a marked degree,
whereas the corresponding product from P. aeruginosa
immunotype 5 contained only a negligible amount of O acetyl groups.5 On the average, it was /0.5 O -acetyl
group per molecule as followed from the 1H NMR
spectrum of the mixture (Fig. 7), which showed
/0.5:1:1 ratios of the integral intensities of the CH3
signals for O -acetyl groups (d 2.1 /2.2), alanine (d 1.54)
and rhamnose (d 1.22 /1.35), respectively. Multiple
signals for the methyl group of Rha suggested that
this sugar residue is O -acetylated.
Tracing connectivities using 1H,1H TOCSY experiments with different mixing times (from 50 to 150 ms)
enabled assignment, starting from the CH3 signals, of all
1
H NMR resonances for five major types of Rha
residues (Table 3). Two of them belong to the nonacetylated Rha residues of core glycoforms 1 and 2 (dH1
4.81 and 5.15), whose contribution to the total rhamnose was /50 and 15%, respectively. Three others were
from mono-O -acetylated Rha residues of core glycoform 1 with an O -acetyl group at position 2, 3, or 4
(/10% each), as followed from a low-field displacement
by 1.15, 1.21 and 1.44 ppm of the signals for H-2, H-3,
and H-4, respectively, as compared with their positions
in non-acetylated Rha (Table 3). The remaining minor
CH3 signals (B/5% each) may belong to O -acetylated
Rha residues of core glycoform 2 and/or to di-O acetylated Rha residues of core glycoform 1.
For more exact determination of the maximum
number of the O -acetyl groups and their distribution,
the LPS was degraded with sodium acetate buffer pH
4.2 at 100 8C. ESI FT-MS analysis of the resulting
mixture showed that this treatment caused a more
significant dephosphorylation as compared with the
HOAc treatment but a lesser O-deacetylation. The
unsubstituted core was found to carry up to three Oacetyl groups (Fig. 8(B)), whereas the core with one Oantigen repeating unit bears no or one O- acetyl group
(Fig. 8(A)). Dephosphorylation of the mixture with
aqueous 48% HF caused partial cleavage of rhamnose,
which resulted in a marked increase of the content of
Rha-lacking compounds. The ESI mass spectrum of the
dephosphorylated mixture confirmed that the full unsubstituted core contains up to three O- acetyl groups
and showed that the Rha-lacking core and core with one
O-antigen repeating unit contain no more than one Oacetyl group (Fig. 9).
Therefore, the majority of the O -acetyl groups are
located on the rhamnose residue. This conclusion is in
accordance with our previous finding that O -acetyl
groups are located in the outer core region of the LPS
of a P. aeruginosa cystic fibrosis isolate.11 The exact
position of the O -acetyl groups beyond rhamnose
remains unknown but it can be suggested that, like
those on Rha, they are distributed randomly all over the
sites in the outer core that are accessible for O-acetyl
transferase(s). Recently, a random O-acetylation of a
lateral 6-deoxy-L-talose residue has been reported in the
O-polysaccharide of Aeromonas hydrophila O-34.15
Comparison of the resonance region of the O -acetyl
groups in the 1H NMR spectra showed that the Oacetylation pattern is similar in all P. aeruginosa strains
studied, whereas the degree of O-acetylation varies from
negligible, as in P. aeruginosa immunotype 5,5 to four
and more O -acetyl groups, as in rough cystic fibrosis
isolate, P. aeruginosa 2192.11
1. Experimental
1.1. Bacterial strains, growth and isolation of
lipopolysaccharides
P. aeruginosa O-12, strain 170023, was from the
Hungarian National Collection of Medical Bacteria
(National Institute of Hygiene, Budapest). Cells were
grown in Roux flasks with solid agar medium on
Hottinger broth at 37 8C for 18 h, then washed in
physiological saline, separated by centrifugation,
O.V. Bystrova et al. / Carbohydrate Research 338 (2003) 1895 /1905
1901
Fig. 5. Parts of ESI mass spectra of fractions IHOAc (A) and IIHOAc (B) obtained by mild acid degradation with aqueous 1% HOAc
from the LPS of P. aeruginosa O-12. The shown regions represent all essential ion peaks found in the complete spectrum, except for
peaks for oligomers (n /1 /4) of the O-antigen repeating unit with m/z 707.3, 1396.6, 2085.9 and 2775.2. Mass peak for compound 6
is shown by asterisk.
washed with acetone and dried. LPS was isolated from
dry bacterial cells by extraction with aq 45% phenol (30
min) at 65 /68 8C.16 Cells were removed by centrifugation, the supernatant was dialyzed against distilled
water; nucleic acids were precipitated by acidification
with aq 50% CCl3CO2H to pH 2.5 and removed by
centrifugation. The supernatant was dialyzed against
distilled water and lyophilized.
1.2. Alkaline degradation
LPS (200 mg) was treated with anhyd hydrazine (4 mL)
at 37 8C for 1 h, diluted with cold water, dialyzed
against distilled water and lyophilized. The product was
dissolved in 4 M NaOH (8 mL), flushed with nitrogen
for 1 h with stirring, heated at 100 8C for 9 h, cooled,
acidified with conc HCl to pH 5.5, extracted twice with
1902
O.V. Bystrova et al. / Carbohydrate Research 338 (2003) 1895 /1905
Fig. 6. Structure of the non-O-acetylated glycoform 1 core 6
obtained by mild acid degradation from the LPS of P.
aeruginosa O-12. Abbreviations: Cm, carbamoyl; Etn, ethanolamine, Hep; L-glycero -D-manno -heptose; Kdo, 3-deoxy-Dmanno -oct-2-ulosonic acid. All monosaccharides are in the
pyranose form and have the D configuration unless stated
otherwise.
CH2Cl2, and the aqueous layer desalted by GPC on a
column (40 /2.6 cm) of Sephadex G-50 (S) in 0.1 M
NH4HCO3 buffer (7.91 g NH4HCO3 and 10 mg NaN3
in 1 L water) at 30 mL h1, using a Knauer differential
refractometer for monitoring of the elution. The isolated
oligosaccharide mixture (27.9% of the LPS weight) was
fractionated by HPAEC on a semi-preparative CarboPac PA1 column (250 /9 mm, Dionex) using a linear
gradient of 0.4 /0.6 M NaOAc in 0.1 M NaOH at flow
rate 1 mL min 1 for 90 min. Two milliliter fractions
were collected and analyzed by HPAEC with pulsed
amperometric detection (Dionex) on an analytical
CarboPac PA1 column (250 /4.6 mm) using a linear
gradient of 0.45 /0.65 M NaOAc in 0.1 M NaOH at a
flow rate of 1 mL min1 for 30 min. After desalting on a
column (40 /2.6 cm) of Sephadex G-50 (S), two major
fractions, INaOH and IINaOH, and a minor fraction
IIINaOH, having retention times 12.55, 16.30, and 18.15
min in analytical HPAEC, were isolated in yields 6.8, 6.1
and 2.6% of the initial oligosaccharide mixture weight,
respectively.
1.3. Mild acid degradation
1.3.1. Protocol A. LPS (200 mg) was dissolved in aq 1%
HOAc and heated for 3 h at 100 8C. The precipitate was
removed by centrifugation, and the supernatant was
fractionated by GPC on a column (40 /2.6 cm) of
Sephadex G-50 (S) as described above. A polysaccharide
and two oligosaccharides, IHOAc and IIHOAc, were
isolated in yields 23.2, 20.0 and 10.2% of the LPS
weight. Oligosaccharide fraction IIHOAc was fractionated by HPAEC on a semi-preparative CarboPac
PA1 column using a linear gradient of 0.02 /0.52 M
NaOAc in water at flow rate 1.5 mL min 1 for 140 min.
Three-milliliter fractions were collected and analyzed by
HPAEC on an analytical CarboPac PA1 column
(Dionex) using the same eluant at 1.5 mL min 1 for
30 min; before pulse amperometric detection the eluate
was mixed (3:1) with 1.5 M NaOH. After desalting on
Fig. 7. Part of a 1H NMR spectrum of the products derived by the mild acid degradation from the LPS of P. aeruginosa O-12.
Shown is the region of the CH3 signals. Designations for Rha of glycoform 1 and 2 core are not italicized and italicized, respectively.
O.V. Bystrova et al. / Carbohydrate Research 338 (2003) 1895 /1905
1903
Table 3
1
H NMR data of the rhamnose residue in the products obtained by mild acid degradation with aqueous 1% HOAc from the LPS of
P. aeruginosa O-12 (d )
Sugar residue
H-1
H-2
H-3
H-4
H-5
H-6
Glycoform 1 core
a-L-Rha
a-L-Rha2Ac
a-L-Rha3Ac
a-L-Rha4Ac
4.81
4.84
4.83
4.85
4.02
5.17
4.17
4.07
3.83
4.02
5.04
4.04
3.45
3.50
3.66
4.89
3.75
3.82
3.88
3.91
1.32
1.34
1.35
1.22
Glycoform 2 core
a-L-Rha
5.15
4.04
3.80
3.47
3.99
1.26
The major signals for the OAc groups are at d 2.160, 2.178, 2.182 and 2.185.
Fig. 8. Parts of an ESI mass spectrum of the products obtained by mild acid degradation at pH 4.2 from the LPS of P. aeruginosa
O-12. Parts A and B show mass peaks for the core with one O-antigen repeating unit and the unsubstituted core, respectively. The
spectrum showed also mass peaks for oligomers of the O-antigen repeating unit, which are outside the ranges shown. The major
mass peak for the compound RhaGlc3(GalNAla)Hep(HepCm)anhKdoP3Ac2 is shown by an asterisk.
1904
O.V. Bystrova et al. / Carbohydrate Research 338 (2003) 1895 /1905
Fig. 9. Part of an ESI mass spectrum of the products obtained by mild acid degradation at pH 4.2 from the LPS of P. aeruginosa O12 followed by dephosphorylation with aqueous 48% HF. Major mass peaks at m/z 1511.52, 1365.45 and 2200.82 belong to the
dephosphorylated non-O-acetylated compounds: the unsubstituted core RhaGlc3(GalNAla)Hep(HepCm)anhKdo, the core lacking
Rha, and the core with one O-antigen repeating unit, respectively. Mass peaks for oligomers (n/2 and 3) of the O-antigen repeating
unit are shown by an asterisk.
Sephadex G-50 (S), the product having retention time
17.7 min in analytical HPAEC (a mixture of 6 and Oacetylated 6) was isolated in a yield 3.8% of the fraction
IIHOAc weight.
1.3.2. Protocol B. LPS (200 mg) was dissolved in 0.1 M
sodium acetate buffer pH 4.2 and heated at 100 8C for 2
h. The precipitate was removed by centrifugation, the
supernatant fractionated by GPC on a column (80 /2.6
cm) of Sephadex G-50 (S) in pyridinium acetate buffer
pH 4.5 (4 mL Py and 10 mL HOAc in 1 L water) at 30
mL h 1. Poly- and oligosaccharide fractions were
isolated in yields 33.3 and 22.4% of the LPS weight.
1.4. Dephosphorylation
The oligosaccharide (2 mg) obtained by degradation of
the LPS with 0.1 M sodium acetate buffer pH 4.2
(Protocol B) was dissolved in aq 48% HF (100 mL) and
kept at 4 8C for 48 h, the solution was dried under
diminished pressure, using a cartridge with solid NaOH
to absorb HF, the residue was dissolved in water and
lyophilized.
1.5. NMR spectroscopy
NMR spectra were obtained on a DRX-600 spectrometer (Germany) in 99.96% D2O at pD 7 and 30 8C
using internal acetone (dH 2.225, dC 31.45) or aq 85%
H3PO4 (dP 0.0) as reference. Prior to the measurements,
the samples were lyophilised twice from D2O. Bruker
software XWINNMR 2.6 was used to acquire and process
the data. Mixing times of 100 and 225 ms were used in
TOCSY and ROESY experiments, respectively.
1.6. Mass spectrometry
ESI FT-MS was performed in the negative ion mode
using a Fourier transform ion cyclotron resonance mass
analyser (ApexII, Bruker Daltonics, USA) equipped
with a 7 T actively shielded magnet and an Apollo
electrospray ion source. Samples were dissolved in
30:30:0.01 2-propanol-water-triethylamine at a concentration of /20 ng mL1 and sprayed with a flow rate of
2 mL min 1.
Acknowledgements
This work was supported by grants from the Deutsche
Forschungsgemeinschaft (SFB 470, project B4 for U.Z.;
436 RUS 113/314 for U.Z. and Y.A.K.; LI-448-1 for
B.L.), the Russian Foundation for Basic Research (0204-04005 for Y.A.K.), INTAS (YS 2001/2-6 for O.B.)
and NIH (AI22535 for G.B.P.).
O.V. Bystrova et al. / Carbohydrate Research 338 (2003) 1895 /1905
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